Antibodies binding human collagen II

ABSTRACT

The present invention relates to antibodies against human collagen II, polypeptides and polynucleotides encoding human collagen II antibodies or fragments thereof, and methods of making and using the foregoing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/386,796, filed 27 Sep. 2010, the entire contents of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies against human collagen II,polypeptides and polynucleotides encoding human collagen II antibodiesor fragments thereof, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

Diseases and conditions that cause the destruction of cartilage withinthe joints pose a significant public health concern, particularly inview of the demographics of an aging population. Multiple mechanisms areinvolved in the degradation of articular cartilage in arthritides suchas rheumatoid arthritis (RA) and osteoarthritis (OA). RA is the mostcommon form of inflammatory arthritis, affecting 3% of women and 1% ofmen. OA, a non-inflammatory arthritis, is the most common form of jointdisease, and is second only to cardiovascular disease as a cause ofearly retirement and disability.

Most treatments for joint ailments are generally systemic. Targeting amedication locally to a joint would have several advantages: increasedefficacy, reduced side effects, an improved dosing schedule, and reducedcost of goods.

Current local treatments including glucocorticoids, injectablehyaluronic acid solutions, NSAIDs or other small molecules haverelatively short half lives as well as systemic distribution onceinjected into the joint (Gerwin, et al., Adv Drug Deliv Rev, 58:226-42,2006; Lindenhayn et al., Eur J Clin Chem Biochem, 35:355-63, 1997).Joint retention of a therapeutic can be achived by coupling thetherapeutic to a joint targeting agent (Rothenfluh et al., NatureMaterials 7:248-54, 2008; WO05/097073; U.S. Pat. No. 7,067,144).However, treatments may require intra-articular injection with deliveryvehicles such as liposomes, adding a layer of complexity and possibleabrasion of the articulating surface.

Thus, there is a need to develop additional vehicles for efficientdelivery and subsequent retention of a therapeutic in the joint.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Human scaffolds for the generation of de novo pIX libraries.Residue numbering according to Chothia. CDR sequences are underlined.

FIG. 2. Sequences of the heavy chain variable regions (VH) and lightchain variable regions (VL) of Fabs binding human collagen II. Residuesdiffering from the wild type human scaffold are indicated. X denotes adeleted residue in the sequence when compared to the wild type.

FIG. 3. Cross-reactivity of Fabs against human and rat collagens I andII.

FIG. 4. Binding of anti-collagen II Fabs to human cartilage.

FIG. 5. Retention of anti-collagen II Fabs in osteoarthritic joints.*P<0.05, **P<0.01; comparison to control CNTO 4234. The counts at 10 minand 1 hour post injection for CNTO 3631 were too high to read.

FIG. 6. Retention of anti-collagen II Fabs in osteoarthritic synovialfluid. *P<0.05; **P<0.01; comparison to control CNTO 4234.

SUMMARY OF THE INVENTION

One aspect of the invention is an isolated monoclonal antibody orfragment thereof that binds human collagen II, comprising a heavy chainvariable region (VH region) and a light chain variable region (VLregion), wherein the VH region comprises the heavy chain complementaritydetermining region (CDR) 1, 2 and 3 (HCDR1, HCDR2, and HCDR3) sequencesas shown in

-   -   i. SEQ ID NO:s 8, 14, and 20;    -   ii. SEQ ID NO:s 9, 15, and 21;    -   iii. SEQ ID NO:s 9, 15, and 22;    -   iv. SEQ ID NO:s 9, 15, and 23;    -   v. SEQ ID NO:s 9, 15, and 24;    -   vi. SEQ ID NO:s 9, 15, and 25;    -   vii. SEQ ID NO:s 9, 15, and 26;    -   viii. SEQ ID NO:s 9, 15, and 27;    -   ix. SEQ ID NO:s 9, 15, and 28;    -   x. SEQ ID NO:s 10, 16, and 29;    -   xi. SEQ ID NO:s 11, 17, and 30;    -   xii. SEQ ID NO:s 12, 18, and 31; or    -   xiii. SEQ ID NO:s 13, 19, and 32; and    -   the VL regon comprises the light chain CDR 1, 2 and 3 (LCDR1,        LCDR2, and LCDR3) sequences as shown in    -   xiv. SEQ ID NO:s 33, 42, and 46;    -   xv. SEQ ID NO:s 34, 42, and 47;    -   xvi. SEQ ID NO:s 35, 43, and 48;    -   xvii. SEQ ID NO:s 36, 44, and 49;    -   xviii. SEQ ID NO:s 37, 42, and 50;    -   xix. SEQ ID NO:s 38, 42, and 51;    -   xx. SEQ ID NO:s 35, 44, and 52;    -   xxi. SEQ ID NO:s 39, 42, and 53;    -   xxii. SEQ ID NO:s 40, 45, and 54; or    -   xxiii. SEQ ID NO:s 41, 42, and 55.

Another aspect of the invention is an isolated monoclonal antibody orfragment thereof that bind human collagen II, comprising a VH region anda VL region, wherein the VH region comprises an amino acid sequencehaving a sequence shown in SEQ ID NO:s 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, or 68, and the VL region comprises an amino acidsequence having a sequence shown in SEQ ID NO:s 69, 70, 71, 72, 73, 74,75, 76, 5, or 7.

Another aspect of the invention is an isolated antibody that binds humancollagen II, comprising a VH region and a VL region, wherein the VHregion comprises the HCDR1, HCDR2, and HCDR3 sequences as shown in SEQID NO:s 9, 15, and 28, and the VL regon comprises the LCDR1, LCDR2, andLCDR3 sequences as shown in SEQ ID NO:s 39, 42, and 53.

Another aspect of the invention is an isolated monoclonal antibody orfragment thereof that binds human collagen II, comprising a VH regionand a VL region, wherein the VH region comprises the HCDR1, HCDR2, andHCDR3 sequences as shown in SEQ ID NO:s 11, 17, and 30, and the VL regoncomprises the LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NO:s34, 42, and 47.

Another aspect of the invention is an isolated antibody heavy chainvariable region comprising the amino acid sequence shown in SEQ ID NO:s56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68.

Another aspect of the invention is a isolated antibody light chainvariable region comprising the amino acid sequence shown in SEQ ID NO:s69, 70, 71, 72, 73, 74, 75, or 76.

Another aspect of the invention is isolated polynucleotides encodingantibody heavy chain variable regions and antibody light chain variableregions of the invention.

Another aspect of the invention is a vector comprising at least onepolynucleotide of the invention.

Another aspect of the invention is a host cell comprising the vector ofthe invention.

Another aspect of the invention is a method of making an antibody thatbinds human collagen II, comprising culturing the host cell of theinvention and recovering the antibody produced by the host cell.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which an invention belongs. Although any compositions andmethods similar or equivalent to those described herein can be used inthe practice or testing of the invention, exemplary compositions andmethods are described herein.

The term “antibody” includes whole antibodies and any fragments thereof.Antibody fragments comprise at least a portion of an immunoglobulinmolecule, such as a complementarity determining region (CDR), a variableregion, a constant region, or a framework region from either antibodyheavy or light chain. An antibody may be a Fab, F(ab′), F(ab′)₂, scFv,dsFv, or diabody. An antibody may be a monoclonal antibody (mAb),chimeric, humanized, or human antibody, dimeric, tetrameric ormultimeric. Structures of the above mentioned antibody fragments, andtechniques for the preparation and use of the antibodies and fragmentsthereof are well known in the art (Ausubel, et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY 1987-2001;Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor, N.Y., 1989; Harlow and Lane, Antibodies, aLaboratory Manual, Cold Spring Harbor, N.Y., 1989; Colligan, et al.,ed., Current Protocols in Immunology, John Wiley & Sons, Inc., NY1994-2001; Colligan et al., Current Protocols in Protein Science, JohnWiley & Sons, NY, N.Y., 1997-2001; Kohler et al., Nature, 256:495-497,1975; Queen et al., Proc Natl Acad Sci, 86:10029-33, 1989; U.S. Pat. No.4,816,567). For example, murine mAbs can be made by the hybridoma methodof Kohler et al., Nature 256:495-497, 1975. Chimeric mAbs can beprepared by the method disclosed in U.S. Pat. No. 4,816,567.Human-adapted mAbs having CDRs derived from a non-human donorimmunoglobulin (typically murine) and the remainingimmunoglobulin-derived parts of the molecule being derived from one ormore human immunoglobulins can be prepared by techniques known to thoseskilled in the art such as that disclosed in U.S. Pat. No. 5,225,539.Human framework sequences useful for human-adaptation can be selectedfrom relevant databases by those skilled in the art. Optionally,human-adapted mAbs can be further modified by incorporating alteredframework support residues to preserve binding affinity by techniquessuch as those disclosed in Queen et al., Proc. Natl. Acad. Sci. (USA),86:10029-10032, 1989 and Hodgson et al., Bio/Technology, 9:421, 1991.

Fully human mAbs lacking any non-human sequences can be prepared fromhuman immunoglobulin transgenic mice by techniques referenced in, e.g.,Lonberg et al., Nature 368:856-859, 1994; Fishwild et al., NatureBiotechnology 14:845-851, 1996; and Mendez et al., Nature Genetics15:146-156, 1997. Human mAbs can also be prepared and optimized fromphage display libraries by techniques referenced in, e.g., Knappik etal., J. Mol. Biol. 296:57-86, 2000; and Krebs et al., J. Immunol. Meth.254:67-84 2001). Fragments of antibodies e.g., Fab, F(ab′)2, Fd, and dAbfragments may be produced by cleavage of the antibodies or byrecombinant engineering. For example, Fab and F(ab′)2 fragments may begenerated by treating the antibodies with an enzyme such as pepsin.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD,IgE, IgG and IgM, depending on the heavy chain constant domain aminoacid sequence. IgA and IgG are further sub-classified as the isotypesIgA₁, IgA₂, IgG₁, IgG₂, IgG₃ and IgG₄.

An antibody variable region consists of a “framework” region interruptedby three “antigen-binding sites”. The antigen-binding sites are definedusing various terms: (i) Complementarity Determining Regions (CDRs),three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1,LCDR2, LCDR3), are based on sequence variability (Wu and Kabat, J. Exp.Med. 132:211-250, 1970; Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991). (ii) “Hypervariableregions”, “HVR”, or “HV”, three in the VH (H1, H2, H3) and three in theVL (L1, L2, L3), refer to the regions of an antibody variable domainswhich are hypervariable in structure as defined by Chothia and Lesk(Chothia and Lesk, Mol. Biol. 196:901-917, 1987). Other terms include“IMGT-CDRs” (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003) and“Specificity Determining Residue Usage” (SDRU) (Almagro, Mol. Recognit.17:132-143, 2004). The International ImMunoGeneTics (IMGT) database(http://www_imgt_org) provides a standardized numbering and definitionof antigen-binding sites. The correspondence between CDRs, HVs and IMGTdelineations is described in Lefranc et al., Dev. Comparat. Immunol.27:55-77, 2003.

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those defined to be antigen-binding site. Theframework is typically divided into four regions, FR1, FR2, FR3, andFR3, which form a scaffold for the three antigen-binding sites in eachvariable region. Because the antigen-binding site can be defined byvarious terms as described above, the exact amino acid sequence of aframework depends on how the antigen-binding site was defined.

The term “antibody that binds human collagen II” as used herein refersto an antibody that binds human collagen II with an EC50 of 1 μg/ml orless in an ELISA assay using plates coated with 10 μg/mL of humancollagen II according to the method described in Example 2.

The term “human collagen II” or huColII as used herein refers to humantype II collagen isolated from cartilage. Human collagen II issynthesized as procollagen alpha Col2A1 chains (SEQ ID NO: 79). Theprocollagen molecule is secreted into the extracellular matrix where itforms fibrils. Fibril formation is accompanied by the removal of the C-and N-propeptides by specific proteinases. Processing of the fibrillarhucolII is well known.

The term “vector” means a polynucleotide capable of being duplicatedwithin a biological system or that can be moved between such systems.Vector polynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than 50 amino acids may bereferred to as “peptides”.

Conventional one and three-letter amino acid codes are used herein asfollows:

Amino acid Three-letter code One-letter code Alanine ala A Arginine argR Asparagine asn N Aspartate asp D Cysteine cys C Glutamate glu EGlutamine gln Q Glycine gly G Histidine his H Isoleucine ile I Leucineleu L Lysine lys K Methionine met M Phenylalanine phe F Proline pro PSerine ser S Threonine thr T Tryptophan trp W Tyrosine tyr Y Valine valVCompositions of Matter

The present invention provides monoclonal antibodies that bind humancollagen II. These antibodies are useful as research reagents,diagnostic reagents, and vehicles for delivering a therapeutic agent forexample to a joint.

The invention provides novel antigen-binding sites and immunoglobulinchains derived from human immunoglobulin gene libraries.

One embodiment of the invention is an isolated monoclonal antibody orfragment thereof that binds human collagen II, comprising a heavy chainvariable region (VH region) and a light chain variable region (VLregion), wherein the VH region comprises the heavy chain complementaritydetermining region (CDR) 1, 2 and 3 (HCDR1, HCDR2, and HCDR3) sequencesand the VL region comprises the light chain complementarity determiningregion (CDR) 1, 2 and 3 (LCDR1, LCDR2, and LCDR3) sequences as shownTable 1.

TABLE 1 SEQ ID NO: Fab ID VH ID VL ID HCDR1 HCDR2 HCDR3 LCDR1 LCDR2LCDR3 VH VL Fab 169-76 VH169-76 VL169-76 8 14 20 33 42 46 56 69Fab169-C2 VH169-C2 VLL6 9 15 21 34 42 47 57 5 Fab169-22 VH169-22VL169-22 9 15 22 35 43 48 58 70 Fab169-34 VH169-34 VL169-34 9 15 23 3644 49 59 71 Fab169-45 VH169-45 VL169-45 9 15 24 37 42 50 60 72Fab169-G11 VH169-G11 VLL6 9 15 25 34 42 47 61 5 Fab169-64 VH169-64VL169-64 9 15 26 38 42 51 62 73 Fab169-47 VH169-47 VL169-47 9 15 27 3544 52 63 74 Fab169-31 VH169-31 VL169-31 9 15 28 39 42 53 64 75 Fab323-G9VH323-G9 VLB3 10 16 29 40 45 54 65 7 Fab323-G1 VH323-G1 VLL6 11 17 30 3442 47 66 5 Fab323-B3 VH323-B3 VLB3 12 18 31 40 45 54 67 7 Fab551-3VH551-3 VL551-3 13 19 32 41 42 55 68 76

Antibodies having conservative substitutions in the heavy and lightchain sequences shown in Table 1 (SEQ ID NO:s 8-76) are encompassedwithin the scope of the invention. The conservative substitution mayreside in the framework regions, or in antigen-binding sites, as longthey do not adversely affect the properties of the antibody.Substitutions may be made to improve antibody properties, for examplestability or affinity. Conservative substitutions will produce moleculeshaving functional and chemical characteristics similar to thosemolecules into which such modifications are made. Exemplary amino acidsubstitutions are shown in Table 2. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for alanine scanning mutagenesis (MacLennan et al.,Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv.Biophys. 35:1-24, 1998). Amino acid substitutions can be done forexample by PCR mutagenesis (U.S. Pat. No. 4,683,195). Libraries ofvariants can be generated using well known methods, for example usingrandom (NNK) or non-random codons, for example DVK codons, which encode11 amino acids (ACDEGKNRSYW). The resulting variants can becharacterized for their binding to human collagen II as described inExamples, or for other properties such as stability using well knownmethods.

TABLE 2 Original residue Exemplary substitutions Ala (A) Val, Leu, IleArg (R) Lys, Gln, Asn Asn (N) Gln Asp (D) Glu Cys (C) Ser, Ala Gln (Q)Asn Gly (G) Pro, Ala His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met,Ala, Phe Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M)Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala, Tyr Pro (P) Ala Ser (S) Thr,Ala, Cys Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V)Ile, Met, Leu, Phe, Ala

In other embodiments, the invention provides an isolated monoclonalantibody or fragment thereof that bind human collagen II, comprising aVH region and a VL region, wherein the VH region comprises an amino acidsequence having a sequence shown in SEQ ID NO:s 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, or 68, and the VL region comprises an amino acidsequence having a sequence shown in SEQ ID NO:s 69, 70, 71, 72, 73, 74,75, 76, 5, or 7.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy and one from a light chain, a skilledartisan will recognize that alternative embodiments may comprise singleheavy or light chain variable regions. The single variable region can beused to screen for a second variable region capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to human collagen II. The screening may be accomplished by phagedisplay screening methods using for example hierarchical dualcombinatorial approach disclosed in Intl. Publ. No. WO92/01047. In thisapproach, an individual colony containing either a H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H), and the resulting two-chain specific antigen-binding domain isselected in accordance with phage display techniques as described.

In another aspect, the invention provides isolated antibody heavy chainsand light chains comprising the amino acid sequences shown in SEQ IDNO:s 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 for heavychains and SEQ ID NO:s 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79for light chains.

Another aspect of the invention is isolated polynucleotides encoding anyof the antibodies of the invention or their complement. Certainexemplary polynucleotides are disclosed herein, however, otherpolynucleotides which, given the degeneracy of the genetic code or codonpreferences in a given expression system, encode the antibodies of theinvention are also within the scope of the invention. Polynucleotidesencoding antibodies of the invention are prepared by well known methods.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and chemical gene synthesis.

Exemplary antibodies of the invention may be of the IgG, IgD, IgE, IgAor IgM isotypes. Additionally, the antibodies of the invention can bepost-translationally modified by processes such as glycosylation,isomerization, deglycosylation or non-naturally occurring covalentmodification such as the addition of polyethylene glycol (PEG) moieties(pegylation) and lipidation. Such modifications may occur in vivo or invitro. For example, the antibodies of the invention can be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation can be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction. (Deckert et al., Int. J. Cancer 87:382-390, 2000; Knight etal., Platelets 15:409-418, 2004; Leong et al., Cytokine 16:106-119,2001; Yang et al., Protein Eng. 16:761-770, 2003).

Pharmacokinetic properties of the antibodies of the invention can beenhanced through Fc modifications by techniques known to those skilledin the art. The “Fc” of an antibody is not involved directly in bindingof an antibody to an antigen, but exhibits various effector functions.An antibody “Fc” is a term well known and is defined on the basis ofpapain cleavage of antibodies. The Fc of an antibody is directlyinvolved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity) based on complement activation, Clqbinding and Fc receptor binding. Complement activation (CDC) isinitiated by binding of complement factor Clq to the Fc of most IgGantibody subclasses. While the influence of an antibody on thecomplement system is dependent on certain conditions, binding to Clq iscaused by defined binding sites in the Fc. Such binding sites are knownin the state of the art and described by, e.g., Boakle et al., Nature282: 742-43, 1979; Lukas et al., J. Immunol. 127: 2555-60, 1981;Brunhouse and Cebra, Mol. Immunol. 16: 907-17, 1979; Burton et al.,Nature 288:338-44, 1980; Thommesen et al., Mol. Immunol. 37: 995-1004,2000; Idusogie et al., J. Immunol. 164:4178-84, 2000; Hezareh et al., J.Virology 75:12161-68, 2001; Morgan et al., Immunology 86:319-24, 1995;EP 0307434. Such binding sites are, e.g., L234, L235, D270, N297, E318,K320, K322, P331, and P329 (numbering according to EU index of Kabat).Antibodies of subclass IgG1, IgG2 and IgG3 usually show complementactivation and Clq binding, whereas IgG4 does not activate thecomplement system and does not bind Clq.

The antibodies of the invention are characterized in that the constantchains are of human origin. Such constant chains are well known anddescribed, e.g., by Kabat (see e.g. Johnson and Wu, Nuc Acids Res. 28,214-18, 2000). For example, a useful human heavy chain constant regioncomprises SEQ ID NO: 77. For example, a useful human light chainconstant region comprises an amino acid sequence of a kappa-light chainconstant region of SEQ ID NO: 78.

The antibodies of the invention may bind human collagen II with a K_(d)less than or equal to about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹ or10⁻¹² M. The affinity of an antibody to human collagen II can bedetermined experimentally using any suitable method. Such methods mayutilize Biacore or KinExA instrumentation, ELISA or competitive bindingassays known to those skilled in the art.

Another embodiment of the invention is a vector comprising at least onepolynucleotide of the invention. The heavy and light chain variabledomains of the invention are combined with sequences of promoter,translation initiation, constant region, 3′ untranslated region,polyadenylation, and transcription termination to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form asingle host cell expressing both chains.

Another embodiment of the invention is a host cell comprising a vectorof the invention. Such host cells may be eukaryotic cells, bacterialcells, plant cells or archeal cells. Exemplary eukaryotic cells may beof mammalian, insect, avian or other animal origins. Mammalianeukaryotic cells include immortalized cell lines such as hybridomas ormyeloma cell lines such as SP2/0 (American Type Culture Collection(ATCC), Manassas, Va., CRL-1581), NSO (European Collection of CellCultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO(ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. Anexemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Otheruseful cell lines include those derived from Chinese Hamster Ovary (CHO)cells such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1(ATCC CRL-61) or DG44.

Another embodiment of the invention is a method of making an antibodybinding human collagen II comprising culturing a host cell of theinvention and recovering the antibody produced by the host cell. Methodsof making antibodies and purifying them are well known in the art.

Uses of the Invention

The antibodies of the invention are useful as research agents, and asdelivery agents of therapeutic molecules to sites expressing humancollagen II, such as a joint.

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the local delivery and jointretention of anti-inflammatory proteins and therapeutic molecules.

Therapeutic molecules may be coupled to the antibodies of the inventionfor improved joint retention. Therapeutic molecules may be proteins orchemical compounds. Exemplary therapeutic molecules are growth factors,cytokines and anti-inflammatory agents, proteins that induce growth andrepair of collagen, as well as small molecules inhibiting proteolyticdestriuction of joint tissue. Therapeutic proteins may be coupled to theanti-collagen antibodies of the invention or fragments thereof bygenerating fusion proteins using well known recombinant methods. Forexample, the N-terminus of the therapeutic protein may be directlylinked to the C-terminus of an antibody of the invention via an amidebond or a peptide linker. Exemplary fusion constructs are described ine.g. U.S. Pat. No. 5,116,964, U.S. Pat. No. 5,709,859, Intl. Publ. Nos.WO04/002417 and WO05/081687. Therapeutic molecules may also be coupledto the antibodies of the invention using chemical crosslinking wellknown in the art, for example using hydrazone or semicarbazone linkage.

The antibodies of the invention with optionally coupled therapeuticmolecule may be prepared as pharmaceutical compositions containing aneffective amount of the therapeutic molecule as an active ingredient ina pharmaceutically acceptable carrier. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the active compoundis administered. Such pharmaceutical vehicles can be liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. For example, 0.4% saline and 0.3% glycine can be used.These solutions are sterile and generally free of particulate matter.They may be sterilized by conventional, well-known sterilizationtechniques (e.g., filtration). The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the agent of the invention in such pharmaceuticalformulation can vary widely, i.e., from less than about 0.5%, usually ator at least about 1% to as much as 15 or 20% by weight and will beselected primarily based on required dose, fluid volumes, viscosities,etc., according to the particular mode of administration selected.Methods for preparing parenterally administrable compositions are wellknown and are described in more detail in, for example, “Remington'sPharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins and proteinpreparations and art-known lyophilization and reconstitution techniquescan be employed.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

EXAMPLE 1 Identification of Collagen II Binding mAbs

Human collagen II-binding Fabs were selected from de novo pIX phagedisplay libraries (Shi et al., J. Mol. Biol. 397:385-396, 2010;WO2009085462A1; U.S. Pat. Appl. No. US20100021477). The libraries weregenerated by diversifying human germline VH genes IGHV1-69*01,IGHV3-23*01, and IGHV5-51*01, and human germline VLkappa genes 012(IGKV1-39*01), L6 (IGKV3-11*01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01).To assemble complete VH and VL domains, the IGHV genes were recombinedwith the human IGHJ-4 minigene via the H3 loop, and the IGKV genes wererecombined with the IGKJ-1 minigene. The positions in the heavy andlight chain variable regions around H1, H2, L1, L2 and L3 loopscorresponding to positions identified to be frequently in contact withprotein and peptide antigens were chosen for diversification. Sequencediversity at selected positions was limited to residues occurring ateach position in the IGHV or IGLV germline gene families of therespective IGHV or IGLV genes. Diversity at the H3 loop was generated byutilizing short to mid-sized synthetic loops of lengths 7-14 aminoacids. The amino acid distribution at H3 was designed to mimic theobserved variation of amino acids in human antibodies. Library design isdetailed in Shi et al., J. Mol. Biol. 397:385-396, 2010. Diversity inthe generated libraries for VH H1 and H2 is shown in Table 4, for H3 inTable 4, and for VL L1, L2 and L3 in Table 5. The scaffolds utilized togenerate libraries were named according to their human VH and VLgermline gene origin. Sequences of the constructed VH and VL scaffoldsused for diversification are shown in SEQ ID NO:s 1-7. (3-23: SEQ ID NO:1; 1-69: SEQ ID NO: 2; 5-51: SEQ ID NO: 3; 012: SEQ ID NO: 4; L6: SEQ IDNO: 5; L27: SEQ ID NO: 6; B3: SEQ ID NO: 7) (FIG. 1). The three heavychain libraries were combined with the four germline light chains orgermline light chain libraries to generate 24 unique VH:VL combinationsfor screening.

TABLE 3 Scaffold Loop Position* 3-23 1-69 5-51 H1 31 SDNT S SNT 32 Y Y Y33 AGW AG W 34 M I I 35 SH S SG H2 50 VANG GW IR 51 I I I 52 SNKW IS YD52a YSGQ PA P 53 SD IY GS 54 G FN D 55 SG G S 56 S T DY *Residuenumbering according to KabatThe libraries were panned using purified human collagen II (Chondrex,#2015, treated with 3M guanidine, DEAE-cellulose and Na₂HPO, 0.5 mg/mLsolution in 0.05M acetic acid) coated on a Maxisorp plate (Nunc) at 10μg/mL in 1× collagen dilution buffer (Chondrex). The libraries wereblocked for one hour at room temperature with 3% milk in TBST. 100 μl ofeach library was combined by heavy chain to generate the 6 HV:HL librarypairs, and applied to pre-blocked (1 hour in 3% milk in TBST) humancollagen II coated plates. After 1 h incubation, the wells were washedin TBST and in PBS five times in each. 200 μL of MC1061 F′cells (OD₆₀₀˜1.0) were added onto the wells for 30 minutes at 37° C., after whichthe infected cells were plated on 2xYT (Carb/Glu) plates and

TABLE 4 H3 length Codon Configuration² 7 A, (NNS) × 3, F/L, D, Y 8 A,(NNS) × 4, F/L, D, Y 9 A, B × 5, F/L, D, Y 10 A, B × 6, F/L, D, Y 11 A,B × 6, (B + BY) × 1, F/L, D, Y 12 A, B × 4, (B + BY) × 4, F/L, D, Y 13A, B × 6, (B + BY) × 3, F/L, D, Y 14 A, B × 6, (B + BY) × 4, F/L, D, YNumbering according to Kabat and co-workers. ²Codon base compositions[A-C-G-T] A: position 1 = [15-15-68-2] position 2 = [43-11-33-13]position 3 = [2-29-24-45] B: position 1 = [19-14-41-26] position 2 =[28-25-33-14] position 3 = [0-34-36-30] B + BY: Mixed primer set at thisposition. BY contains a tyrosine codon (TAT) sequentially replacing theB codon in these positions and is mixed at a ratio of 1:7 with a primercontaining the B codon. Therefore, for CDR length 11 with one (B + BY)codon, two primers were used. One had the TAT codon at this position andthe second had the B codon and these were mixed at a ratio of 1:7. ForCDR length 13, the three (B + BY) codons required 4 primers, one withthe B codon and three with the TAT codon in each of three positions andthe B codon in the remaining 2 positions. In this case, the TAT primerswere mixed at a ratio of 1:1:1:7 with the full B codon primer.Similarly, CDR-12 and CDR-14 were prepared with 5 primers.placed at 37° C. overnight. The colonies that had grown overnight werescraped off the plates in 2 mL per plate of 2xYT (Carb/Glu/20%glycerol). 50 ml of re-suspended bacteria was used to inoculate a 20 mLculture in 2xYT (Carb) and the remainder of the bacteria were frozen.The cultures were grown at 37° C. to an OD₆₀₀ 0.5-1.0, after which 1 mLof helper phage VCSM13 (Stratagene, Cat. No. 200251) was added to theculture at a multiplicity of infection of approximately 10:1 andincubation was carried out for 30 minutes at 37° C. without shaking.Kanamycin and IPTG were added the the culture and it was grown at 30° C.overnight. Phage was precipitated with 2% PEG/0.25M NaCl andre-suspended in 1 ml of PBS. One fifth of the PEG-precipitated phage wasused to initiate the next round of panning and the remaining phage werestored at −20° C. The selection cycle was repeated four times. After thelast selection cycle, the colonies were scraped off in 2 mL 2xYTCarb/Glu/20% glycerol and 100 μL of the cell suspension was used toisolate plasmid DNA. The pIX was excised by NheI/SpeI digestion andself-ligation of the isolated DNA. After ligation, the DNA waselectroporated into electrocompetent MC1061 F′ cells and plated forsingle colonies on 2xYT Carb/Glu.

TABLE 5 Scaffold Loop Position* O12 L6 A27 B3 L1 30 SRNAD SRNAD SRNTD L30a — — SNR YSHFA 30e — — — KTNE 31 SNKDG NSKD SNRADH K 32 YHNDWFSAYWDFHSAN YFHQSEK YFHNWDAS L2 50 FYTNKADG ADKGYFTN ADGS WSRDYA L3 91SAYHPD RYSGF YSHA YSHA 92 FIYHNDKGR RHNSL YNDSHIFKG YNDSHIFKG 93 STHNDRGNDKR SNTDGHR SNTDGHR 94 TYLVFSRGP WA TYLVFAS TYLVFAS 96 LWRFYIN WYFLIRWYFLIR WYFLIR *Residue numbering according to Kabat

EXAMPLE 2 Cross-Reactivity of Col II mAbs with Other Collagens

The human collagen II binding Fabs obtained from the initial panningwere screened for cross-reactivity with human collagens I, IV and V, andrat collagens I and II.

Preparation of Fab Lysates.

Colonies were picked from the pIX-excised transformations and grown in2xYT Carb. The next day, 50 μL of the saturated cultures were used toinoculate an expression plate containing 400 μL per well of 2xYT Carband the plate was grown at 37° C. for 6 hours. Fab expression wasinduced with the addition of 1 mM IPTG and the plate was placed at 30°C. overnight. The next day, the induced Fab cultures were spun at 2000rpm for 10 minutes, and the cleared lysate was used in subsequentassays.

ELISA

Maxisorp ELISA plates (Nunc) were coated with 10 μg/ml human collagen I(Chondrex), human collagen II (Chondrex), rat collagen I (Chondrex), ratcollagen II (Chondrex) or with 5 μg/ml human collagen IV (Chemicon) orhuman collagen V (Chemicon), or with anti-Fd (the Binding Site) at 1μg/ml, all in PBS, according to manufacturer's instructions. The wellswere washed three times in TBST and blocked for one hour with 200 μL 3%milk in TBST. 100 μL of the Fab lysate was added onto the wells of thecoated ELISA plates, and incubated 1 hour at room temperature. The wellswere washed three times followed by addition of 100 μL of anti-kappa-HRP(Southern Biotech) at 1:5000 in PBS. The plates were incubated at roomtemperature for one hour, washed three times with TBST, and developedwith BM Chemiluminescence ELISA Substrate (Roche Applied Science). Theclones that bound to human and rat collagen II without binding to humancollagen I, IV or V, or rat collagen I were sequenced and unique cloneswere further characterized. FIG. 3 shows the ELISA data for the 13clones chosen for further characterization. The sequences of the Fabsare shown in FIG. 2 and Table 1.

EXAMPLE 3 Col II mAbs Bind to Human Cartilage

Small-Scale Fab Purification

The Fab expression in Escherichia coli in 2xYT Carb (except thatTurboBroth (Athena ES) was used for expression of Fab 551-3) was inducedwith 1 mM IPTG at 30° C. overnight. Induced bacteria were pelleted 30min 4500 rpm, the cell pellets resuspended in lysis buffer (20 mM Tris,pH 8.5, 350 mM NaCl, 7.5 mM imidazole) with protease inhibitors, andruptured with two passes through a microfluidizer. The cell lysate wasclarified with two spins at 10,000 rpm for 10 minutes. Talon resin(Clontech) was equilibrated with lysis buffer and two mLs were added tothe clarified lysate. Bound Fabs were eluted with two incubations of 5minutes each using elution buffer (150 mM EDTA, 20 mM Tris, pH 8.5) anddialyzed in 20 mM Tris, pH 8.5. The dialyzed Fabs were further purifiedusing a Q-sepharose Fast Flow resin (QFF resin; GE Healthcare), and usedfor experiments.

Cartilage-Binding Assay

Human cartilage was obtained from osteoarthritis patients (NorthlandLaboratories). The cartilage was first pulverized and stored as asuspension in PBS with protease inhibitors at 4° C. To test forcartilage binding, 2.5 μL of cartilage suspension per data point wasadded to 97.5 μL of TBST with 3% milk and the mixture rotated at roomtemperature for one hour. Binding of Fabs to the cartilage was testedusing a MultiScreen HTS plate (Millipore) using the vacuum manifold.Briefly, 100 μL of the suspension was added into prewetted MultiScreenplate, and vacuum was applied to settle the cartilage onto the wellfilter, after which the wells were washed twice with TBST. 2.4 μg ofeach Fab in 100 μL of PBS was added to the wells and incubated at roomtemperature for 1 hour. The wells were washed three times with TBST, andanti-kappa-HRP (Southern Biotech) was added onto the wells. After onehour incubation, the wells were washed in PBST, the BM ChemiluminescenceELISA Substrate (Roche Applied Science) was added, and the resultantsuspension of cartilage fragments was transferred to a black-well ELISAplate for luminescence detection. All 13 Fabs exhibited cartilagebinding (FIG. 4).

EXAMPLE 4 mAb Affinities to Col II

ELISA

EC50 values were obtained for select Fabs using ELISA assay as describedabove. In the assays, 100 microliters of each Fab was added in aconcentration range between 10 ng/ml-10 μg/ml onto the wells. The EC50values are shown in Table 6.

Biacore

Biacore binding kinetics was performed using standard methods againsthuman and rat collagen II (Table 6b).

TABLE 6a Fab EC50 (ng/ml) 551-3 113.5 323-G1 123.1 169-31 113.1

TABLE 6b human collagen II rat collagen II K_(a) × K_(d) × K_(D) K_(a) ×K_(d) × K_(D) Fab 10⁵ 10⁻³ (nmol) 10⁵ 10⁻³ (nmol) 323-G1 1.84 1.08 62.94 1.39 5 169-31 0.97 5 50 1.05 7.89 75 CNTO 4234 n.a. n.a. >1000 n.a.n.a. >1000

EXAMPLE 5 Anti-Collagen II mAbs are Retained in the Joints In Vivo

Two anti-collagen II Fabs (323-G1 (CNTO 3631) and 169-31 (CNTO 4093) anda control mAb (CNTO 4234) that did not bind to extracellular matrixcomponents were iodinated and injected into the knees of menisectomizedrats to evaluate the effect of mAb binding to collagen on jointresidence time. Fabs were radiolabeled using Na¹²⁵I (Perkin Elmer) andIodo-GEN tubes (Pierce). Free iodine was removed using PD-10 desaltingcolumns (GE Healthcare) and the Fabs were concentrated to 2.2 mg/mLusing Amicon Ultra centrifugal filter devices (Millipore; 10,000 MWCO).After iodination, the Fabs were tested for binding of collagen II, andno significant impairment due to iodination was observed.

32 male Sprague-Dawley rats underwent meniscus transection on the rightand left hind limb. The rats were anesthetized with isofluraneanesthesia 2-4%, and the medial collateral ligament was transected andthe meniscus cut to simulate a tear injury. Joint damage resemblingosteoarthritis developed over the next three weeks. Twenty-one daysafter surgery, intra-articular injections of ¹²⁵I-labeled Fabs wereperformed. Animals received 11 μg of test article through 5 μlintra-articular injections into the right and left knee joint. Animalswere euthanized at four time points (10 min, 1 hr, 6 hr and 24 hrspost-injection) and synovial fluid lavage and the knee joints werecollected from bot knees. Radioactivity was measured from each sample.The counts per minute (cpm) were compared to the values of thenon-injected dose of each respective Fab to calculate the % retention(e.g. as percent of injected dose). 323-G1 (CNTO 3631) and 169-31 (CNTO4093) were retained both in the knee joint (FIG. 5) and synovial fluid(FIG. 6) longer than the control antibody. Approximately 60% and 32% ofFab 323-G1 and 60% and 18% of Fab 169-31 were retained in the knee(combined joint and synovial retention) after 6 and 24 hours postinjection, respectively. The control Fab was retained at 17% and 5%.Thus, the antibodies of the invention exhibited increased jointretention due to their binding to a resident joint protein.

1. An isolated monoclonal antibody or fragment thereof that binds humancollagen II, selected from the group consisting of: i. a VH region and aVL region, wherein the VH region comprises the HCDRI, HCDR2, and HCDR3sequences as shown in SEQ ID NO: 11, 17, and 30, respectively, and theVL region comprises the LCDRI, LCDR2, and LCDR3 sequences as shown inSEQ ID NO: 34, 42, and 47, respectively, ii. a VH region and a VLregion, wherein the VH region comprises the HCDRI, HCDR2, and HCDR3sequences as shown in SEQ ID NO: 9, 15, and 28, respectively, and the VLregion comprises the LCDRI, LCDR2, and LCDR3 sequences as shown in SEQID NO:s 39, 42, and 53, respectively, and iii. a VH region and a VLregion, wherein the VH region comprises the HCDRI, HCDR2, and HCDR3sequences as shown in SEQ ID NO: 13, 19, and 32, repectively, and the VLregion comprises the LCDRI, LCDR2, and LCDR3 sequences as shown in SEQID NO: 41, 42, and 55, respectively.
 2. An isolated monoclonal antibodyor fragment thereof that bind human collagen II, comprising a VH regionand a VL region, wherein the VH region comprises an amino acid sequencehaving a sequence shown in SEQ ID NO: 64, 66 or 68 and the VL regioncomprises an amino acid sequence having a sequence shown in SEQ ID NO:75, 76, or
 5. 3. The isolated antibody of claim 1, wherein the antibodycomprises a Fab fragment.
 4. An isolated monoclonal antibody or fragmentthereof that binds human collagen II, comprising a heavy chain variableregion comprising the amino acid sequence shown in SEQ ID NO: 64, 66 or68.
 5. An isolated monoclonal antibody or fragment thereof that bindshuman collagen II, comprising a light chain variable region comprisingthe amino acid sequence shown in SEQ ID NO: 75 or 76.